The invention relates to murine monoclonal a tibodies (MAbs), A, B, C and D, which are directed against tumor-associated antigens. The nearly complete nucleotide sequences of the V genes of these MAbs are described, so that the relevant variable domains can be put together to give chimeric MAbs, or “humanized” MAbs are obtained by inserting the hypervariable regions (complementarity determining regions CDR) into a human MAb framework. Antibody constructs of this type can be employed in human therapy and in vivo diagnosis without the disadvantages observed with murine MAbs.
MAb A reacts with antigen 2, MAb B reacts with antigen 11 and MAb C reacts with antigen 7, all of which are described in EP-A2 0,141,079 and are membrane-associated antigens on permanent human tumor cells lines such as the CaLu-1, Chago, Oat 75, PaTuII and Bewo cell line. MAb D is directed against a Vibrio cholerae neuraminidase (VCN)-sensitive epitope on the ganglioside GD2 which is exposed on human melanoma cell lines.
MAbs A to D were generated as described in EP-A2 0,141,079 and isolated by standard methods.
MAb A binds to cells of the granulocyte compartment and to carcinomas of the colon, pancreas and some of the lung and breast, as shown in Tab. 1.
TABLE 1Binding characteristics of MAb AMalignant tissue samplesNumber ofTotalinvestigatedpositivesnumbercolorectal carcinomas:primary carcinomas66liver metastases1516carcinomas of the pancreas68carcinomas of the lung:small-cell12adeno910squamous cell34large-cell12carcinoma of the breast13
MAb B binds to virtually all carcinomas of the gastrointestinal tract and to some ovarian carcinomas and adenocarcinomas of the lung, whereas it does not react with most normal human tissues or reacts only with secretion-containing sites thereon. The binding characteristics are summarized in Tab. 2.
TABLE 2Binding characteristics of MAb BMalignant tissue samplesNumber ofTotalinvestigatedpositivesnumbercolorectal carcinomas:primary66liver metastases910carcinomas of the pancreas56carcinomas of the stomach44carcinomas of the lung:small-cell211adeno910squamous cell212large-cell 4*12carcinoma of the breast29ovarian carcinomas(secretion-containing sites)46carcinomas of the kidneys112*weak, heterogeneous reaction 
MAb C shows a distinct reaction with 70-80% of stage I and II primary tumors of carcinoma of the pancreas (11/14). Like the primary tumors, most grade I and II metastases of carcinoma of the pancreas appear to have a positive reaction (3/4). The type of reaction on these positive tissues indicates that the epitope recognized by MAb C is located in the cytoplasm, on the membrane and in the intercellular space.
Grade III carcinomas of the pancreas do not express the epitope (primary tumor 0/2, metastases 0/5). However, since grade I and II carcinomas of the pancreas comprise up to 95% of carcinomas of the pancreas, MAb C ought to react with 60-70% of all carcinomas of the pancreas.
Another important property of MAb C is its reactivity with the duct system in the chronically inflamed pancreas (pancreatitis 10/13), whereas there is no detectable binding to healthy exocrine and endocrine pancreatic tissue (0/8). Only a minority of the investigated primary tumors of carcinoma of the colon was reactive (4/14), whereas most of the liver metastases of carcinoma of the colon showed distinct binding (7/10).
Cross-reactivities of MAb C are confined to the mucus-producing goblet cells of the colonic mucosa and of the stomach. All the other normal tissue tested, including peripheral blood leukocytes and bone marrow, do not express the mucus-associated epitope recognized by MAb C (see Tab. 3). MAb C recognizes an epitope located on a tumor associated antigen which can be detected at increased levels in patients with gastrointestinal tumors e.g. carcinomas of the pancreas and thus may be used as a tumor marker.
TABLE 3Summary of the formaldehyde-fixed, paraffin-embeddedhuman tissue investigated (indirect immunoperoxidase)with MAb C.Pancreatic tumorsGrade I/IIGrade IIIprimary carcinoma11/140/2liver metastases of carcinoma3/40/5papillary carcinoma1/2cystadenoma0/1Pancreatic tissueducts with changes due topancreatitis10/13normal exo- and endocrine0/8pancreasCarcinomas of the colonprimary tumors4/14liver metastases7/10Normal colonic tissuegoblet cells of the mucosa,9/10luminal partpart of the mucosa facing the muscularis3/10muscularis mucosae0/7tunica muscularis0/7submucosa0/7Carcinomas of the lung8/18(a few tumor cells)Carcinomas of the breast0/3Other normal tissue investigatedlung0/3liver0/5breast0/1Stomachmuscle0/2mucus-prod. cells3/3kidney0/5lymph nodes0/4spleen0/2bone marrow0/1peripheral blood leukocytes0/2connective tissue0/2muscles0/10
MAb D recognizes a VCN-sensitive epitope on the ganglioside GD2 which does not occur on other bovine cerebral gangliosides, including GD3, GM3, GM1, GT1a, GD1b, GD1a and GM4. Using MAb D it was possible to stain all gliomas, meningiomas and neurilemmomas in normal human cerebral tissue. Tab. 4 summarizes the binding properties of MAb D with respect to intracranial tumors.
TABLE 4Number of tumorsType of tumorPositiveTotal numberwell-differentiated gliomas,66grade I-IImalignant gliomas,1010grade III-IVmeningiomas1111neurilemmomas44pineal adenomas05metastases of carcinomas01
The epitope defined by MAb D is also present on neuroblastomas, ganglioneuroblastomas and ganglioneuromas. Tab. 5 shows a summary.
TABLE 5Reactivity of MAb D with neuroblastomas and small round-cell tumors in childrenNumber of tumorsNeuroblastomasPositiveTotal numberGrade I11Grade II66Grade III44ganglioneuroblastomas 3xx3Ewing sarcomas02rhabdomyosarcomas01non-Hodgkin lymphomas  2xxx5xHughes classification xxganglion cells xxxsome positive tumor cells 
Thus, MAb D is suitable for differential diagnosis of neuroblastomas and small round-cell tumors in children.
The specificity of MAb D for two other tumors derived from the neuroectoderm, namely melanomas and small-cell carcinomas of the lung (SCLC), and for unrelated tumors is shown in Tab. 6.
TABLE 6Number of tumorsPositiveTotal numbermelanoma 4*10SCLC 2*11carcinomas of the colon03carcinomas of the breast03non-SCLC:adenocarcinomas03squamous cell carcinomas of03the lunglarge-cell carcinomas03*Weak and heterogeneous staining of a few tumor cells 
The immunoglobulin V genes of MAb A to MAb D were isolated using the methods detailed in the Examples.
The nucleotide and protein sequences are shown in Tables 7 (V gene of MAb A), 8 (V gene of MAb B), 9 (V gene of MAb C) and 10 (V gene of MAb D). The CDRs can be identified therein as described by Kabat and Wu (loc. cit., see Examples).